Development of a high-throughput screen to detect the effects of both pre- and post- biotransformed compounds for enhanced content drug discovery workflows Project Summary This Small Business Innovation Research Phase I project proposes to engineer a panel of autonomously bioluminescent human cell lines for the simultaneous, high-throughput detection of both pre- and post- biotransformed cytotoxicity onsets resulting from drug treatment across multiple tissue types to address the National Institute of General Medical Sciences (NIGMS) request for novel in vivo and in vitro methods for predicting the safety and toxicities of pharmacologic agents. With an average of 10,000 novel molecules that must be screened for each new lead compound developed, and an average of 10 to 15 years of research and development at a cost of up to $1B to manufacture one new drug, pharmaceutical companies must develop new testing regimens that provide more data at a lower cost in order to achieve the economics necessary to remain profitable. Up to 92% of failures for these new compounds at the clinical level are related to cytotoxicity, which often onl manifests during the costly and time consuming process of whole animal testing. This problem could be significantly mitigated by coordinately screening multiple tissue types simultaneously in a high-throughput fashion during upstream tier 1 screening. However, with existing bioluminescent reporter technology, this is simply not possible because the current market of bioluminescent reporter cells being applied toward toxicology screening relies upon a firefly luciferase gene construct that must be provided with a chemical substrate to activate its light emission response, resulting in only marginally informative single time point snapshots of potential toxicological interactions. In contrast, our substrate-free, autobioluminescent reporter cell lines emit light continuously and can modulate the output level of this signal in real time in response to cytotoxic interactions. This provides an uninterrupted stream of visual data over the lifetime of the reporter cell as it interacts and reacts to compound exposure at both its pre- and post-biotransformed states. Furthermore, by leveraging human cells as the sensing platform, our assay provides more accurate and realistic information in regards to bioavailability and a chemical's true effect on individual human health than does the employment of small animal models. With current in vitro screening assays now representing a $1.4B market with a predicted 12% annual growth rate, we believe we possess a product capable of significantly impacting the chemical/drug screening market and, here in particular, advancing our understanding of cytotoxic chemical biotransformations as they pertain to public health and consumer safety.